Project summary. Cerebral pericytes are specialized mural cells that line the entire cerebrovascular capillary bed. The concept that pericytes are contractile and have the capacity control capillary flow dates back to their discovery in the 1890s. Yet, due to a lack of methods to target and manipulate pericytes in vivo, this facet of pericyte biology has remained highly understudied. It is imperative to understand pericyte contractility because many human and animal studies have demonstrated insufficiency in capillary flow during stroke, with aberrant constriction of capillaries being a likely contributor. Modulating the contractile ability of pericytes may represent a valuable therapeutic approach to improve cerebral blood supply during ischemia, and a means to further improve the efficacy of existing clot-removal treatments. Currently, little is known about the signaling pathways involved in pericyte contraction. Recent studies have shown that the vast majority of pericytes that line the brain capillary bed are negative for ?- smooth muscle actin (?-SMA), which is central to actomyosin-based contraction of smooth muscle cells of arterioles. Yet, our preliminary data suggest that these ?-SMA-negative pericytes retain the ability to contract in vivo and can impede capillary flow, pointing to an alternative contractile mechanism. Our central hypothesis is that brain capillary pericytes can contract through dynamic actin cytoskeleton reorganization, rather than actomyosin cross-bridge cycling. We test this hypothesis by combining pharmacology with a novel optogenetic assay to activate individual capillary pericytes in a ?cause and effect? manner both in vivo and ex vivo. In Aim 1, we will test whether drugs that inhibit or promote actin polymerization can alter optogenetically-induced pericyte contraction in the brains of live mice. We will further test these drugs on pericyte contractility in an ex vivo, pressurized arteriole-to- capillary preparation to exclude indirect actions from non-vascular brain cells. In Aim 2, we directly visualize actin polymerization in capillary pericytes in the normal and ischemic brain in vivo. We will express Lifeact-GFP, a novel fluorescent probe for F-actin, specifically in vascular mural cells and examine whether F-actin content increases in pericytes prior to pathological capillary constriction. This project will shed light on capillary pericyte cytoskeletal dynamics and its relation to capillary flow, an aspect of pericyte biology that is highly understudied in the brain microvasculature in vivo. If successful, our findings will help to establish the rationale, methodologies, and mouse models for further investigations of how pericyte cytoskeletal dynamics are involved in capillary flow impairment during stroke and related brain pathologies.